Abstract
An ultrasensitive separation free micro volume bioaffinity assay method is presented. The concept is based on the use of two-photon excited fluorescence and polystyrene microspheres as bioaffinity binding solid phase. Measurements are performed by observing individual microspheres in the diffraction limited focal volume of two-photon excitation. The method is suitable for measuring bioaffinity binding assays directly from the reaction volume without separation and washing steps. Depending on the sensitivity requirements the assay can be scaled. Scaling is achieved by adjusting the amount of solid phase microspheres for the assay. The scalability is demonstrated by measuring standard curves for an immunoassay. As a model assay we chose the human alpha-fetoprotein (AFP) immunometric assay. Operating ranges from 10 pg/ml up to 20 ng/ml and 100 pg/ml up to 400 ng/ml were achieved.
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